The long-term goal of the proposed research is to understand how a novel class of DNA elements called insulators regulates gene expression. Insulators were first identified in Drosophila as chromatin boundary elements that separate neighboring genes and prevent them from influencing each other. Sequences of similar function have also been identified in avian and mammalian species. Examples of Drosophila insulators include the scs and scs elements from the hsp70 locus and the su(Hw) element from the gypsy retrotransposon. An important characteristic of insulator activity is that they repress the function of enhancers located upstream but not downstream from them. Recent genetic evidence indicates that insulator function may represent a general mechanism of gene regulation, especially in complex genetic loci. For example, the Fab-7 and Mcp-1 insulators in the Abd-B regulatory region control the levels of Abd-B expression in parasegment 9 and 11 by specifying interactions between the Abd-B promoter and its tissue-specific enhancers. We have recently devised an enhancer-blocking assay to study insulator function in the Drosophila embryo. The study revealed that insulators can block interactions between a promoter and distal enhancers without affecting their activities per se. It also showed that insulators exhibit enhancer and/or promoter specificity. Using this assay, we have identified a novel insulator (SF-1) in the Antennapedia complex (ANT-C). The unique position of SF-1 between two divergently transcribed homeobox genes (Scr and ftz) suggests that it may regulate enhancer-promoter interactions in the genomic interval by specifying cognate enhancer-promoter interactions. It is not known how SF-1 affect gene expression in ANT-C or how insulators block enhancer-promoter interaction in general. This application proposes to study two insulator elements, focusing on two different aspects of their functions: 1) SF-1 will be analyzed for its regulatory roles in specifying Scr enhancer- promoter interaction in transgenic minigene complexes. Protein components of the insulator function of SF-1 will be identified through genetic screens. 2) The su(Hw) element will be examined for its enhancer-blocking mechanism, including its blocking specificity and its ability to form topologically separate chromatin loop domains. Understanding how insulators specify enhancer-promoter interactions and regulate gene activity, especially in the conserved homeotic gene complexes, will not only aid our understanding of the diverse mechanisms of gene regulation, but should also shed light on an important developmental process that specifies body plans from fly to man.